Nuclear Membrane Dynamics in Rhabdovirus-infected Cells

Members of the family Rhabdoviridae are enveloped viruses with monopartite, minus-sense, single-stranded RNA genomes that include some of the greatest threats to human, animal, and plant health. My research group is using Sonchus yellow net virus (SYNV) and Potato yellow dwarf virus (PYDV) as paradigms for cell biology studies of rhabdoviruses that replicate in nuclei of infected cells. We have identified novel cellular events that underlie rhabdovirus-plant interactions, including alterations in the nuclear membranes of infected cells, which we have recently reported. Further, we have identified an unprecedented interaction involving the SYNVencoded matrix protein, putative RNA-silencing suppressor protein and nucleocapsid protein, as well as the nuclear pore complex in host cells. These observations, and future studies, require novel approaches in order to resolve the underlying molecular and cellular basis. Therefore, in Objective 1 of this proposal we will generate a series of double-transgenic Nicotiana benthamiana plants that express SYNV -encoded proteins or fluorescent reporter proteins under the control of an inducible promoter, as well as GFP targeted to endomembranes under the control of a constitutive promoter. In Objective 2, we will develop a second set of double-transgenic plants to test the hypothesis that infection by rhabdoviruses induces relocalization of the nuclear membrane. Markers targeted to the nuclear pore complex, inner nuclear membrane or chromatin will be expressed as fusions to the monomeric orange fluorescent protein in the same GFP-expressing plants used in objective 1. In Objective 3, we will test nucleoporins for their ability to interact with purified P protein.